Programs that control disease-transmitting vectors often use insecticides, both chemical and biological. Thus Bacillus and Lysinibacillus sp. have been used worldwide for over four decades for successful control of mosquitoes and blackflies. But a novel strain, Clostridium bifermentans (Cb) malaysia, the most toxic bioinsecticide to Anopheles has not been used, even though being an anaerobe; it is much easier to culture with minimal equipment. The lack of knowledge of what the toxins are in Cb malaysia prevents its utilization. Thus our long-term goal is to understand Cb malaysia mosquitocidal action, facilitating its use for the control of anopheles larval mosquitoes, thereby attenuating adult populations and malarial transmission. The overall objective of the current proposal is to identify the anopheline active toxins and elucidate their mechanism of action. To identify the mosquitocidal toxins involved, we generated a loss of function Cb malaysia mutant. Genomes of this mutant, Cbm-77, together with that of wild-type Cb malaysia and of the non-mosquitocidal type strain Cb were sequenced. The data obtained demonstrated that a loss of a megaplasmid in the Cbm-77 mutant, or its absence in Cb, results in total loss of mosquitocidal activity. Hence, we hypothesize the mosquitocidal activity is encoded by the plasmid, and specific genes in this plasmid are responsible for high Cb malaysia toxicity to anophelines. Mass spectrometry data confirmed that the anopheline-toxic proteins were encoded by the megaplasmid, and these proteins form a complex. The plasmid has two toxin encoding loci, cry and ctox; the former consists of a single operon, while the second locus has a cmp (clostridal mosquitocidal protein) operon and two additional genes, p47 and ha41. Therefore in the first aim we will validate our preliminary results by: a) identifying the critical anopheline active toxins in the ctox locus. Sine this locus encodes proteins that are similar to those produced by other Clostridium strains we hypothesize that the HA41 protein is involved in midgut membrane binding, while the CMP, which has an endopeptidase motif, is the active toxin that could cleave SNARE complexes required for vesicular release. Preliminary evidence supports both hypotheses. We will test these hypotheses in the next two aims: b). Characterize the binding of the Cb malaysia toxins to midgut membranes; and c). Define the intracellular target site for the Cb malaysia toxins. Completion of this proposal will show the utility of this novel bacterium because of its ease of use. Its novel mode of toxin action also complements that of toxins from B.t. israelensis and L. sphaericus, facilitating its use as a biocontrol agent for anopheline control. Its novel mechanism of toxicity would broaden the spectrum of available anopheline-active bioinsecticides, and will also help attenuate resistance development.